Detection of Immunogenic Proteins from Anopheles Sundaicussalivary

Revista da Sociedade Brasileira de Medicina Tropical 48(4):410-416, Jul-Aug, 2015 Major Article http://dx.doi.org/10.1590/0037-8682-0185-2015

Detection of immunogenic proteins from Anopheles sundaicus salivary glands

Yunita Armiyanti[1], Mohammad Mirza Nuryady[2], Renam Putra Ariﬁ anto[2], Elisa Nurmariana[2], Kartika Senjarini[2], Loeki Enggar Fitri[3] and Teguh Wahju Sardjono[3]

[1]. Department of Parasitology, Faculty of Medicine, Jember University, Jember, Indonesia. [2]. Department of Biology, Faculty of Mathematic and Natural Sciences, Jember University, Jember, Indonesia. [3]. Department of Parasitology, Faculty of Medicine, University of Brawijaya, Malang, Indonesia.

ABSTRACT Introduction: The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of speciﬁ c IgG antibodies in the host, which can be used as speciﬁ c biomarkers of exposure to Anopheles sundaicus. The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes. Methods: IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting. Results: Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls. Conclusions: These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines. Keywords: Anopheles sundaicus. Salivary glands. Immunogenic proteins. Antibody.

in coastal area of Java and the Sumatra islands. The distribution INTRODUCTION of Anopheles sundaicus also includes countries in Southeast Asia and India(2) (3). Malaria is a mosquito-borne disease that has major health The important role of Anopheles mosquitoes as malaria implications worldwide, with an estimated 207 million vectors is supported by the presence of salivary glands in individuals affected by malaria and 627,000 malaria-related female Anopheles mosquitoes. Anopheles mosquito salivary deaths reported. In 2013, 104 countries and territories, including glands secrete substances that can enhance the transmission of Indonesia, had malaria, which was considered endemic; an Plasmodium(4). These substances inhibit hemostatic processes estimated 3.4 billion people are at risk of contracting malaria(1). and modulate the host immune response (immunosuppressive) Malaria is caused by ﬁ ve species of parasites from the at site of biting, allowing the mosquito to feed on blood genus Plasmodium and naturally spread from one person to successfully(5) (6). These changes at the site of the bite, owing to another by female Anopheles mosquitoes. There are about 400 the effects of salivary substances, would beneﬁ t the pathogen, different species of Anopheles mosquitoes, but only 30 of these permitting infection without any resistance and thus enhancing are vectors of major importance(1). Anopheles sundaicus is one infectivity in the vertebrate host(7). of the most important malaria vectors in Indonesia, particularly Injection of saliva into the host’s skin also induces the production of antibodies against salivary proteins(8). The immunogenicity of salivary proteins has been demonstrated Corresponding author: Yunita Armiyanti. Department of Parasitology/ in previous studies, which showed that salivary proteins can Faculty of Medicine/Jember University. Jember 68121, Indonesia. induce the host immune response by producing immunoglobulin Phone: 62 857 3248-2300 email: [email protected] G (IgG) and immunoglobulin E (IgE) via hypersensitivity (9) Received 19 June 2015 reactions . The presence of a specific IgG antibody Accepted 15 July 2015 response against whole saliva extracts (WSEs) of Anopheles

410 Armiyanti Y et al. - Anopheles immunogenic protein mosquitoes can be measured by enzyme-linked immunosorbent glands were dissected using fi ne entomological needles under a assays (ELISAs) in children and adults exposed to mosquitoes stereoscopic microscope at 4× magnifi cation and collected into bites(10) (11). Additional studies have shown that the IgG response a microcentrifuge tube containing a small amount of phosphate- can recognize specifi c salivary proteins(12) (13). Therefore, salivary buffered saline [(PBS); pH 7.2] and phenylmethylsulfonyl proteins (e.g., gSG6) that are able to generate specifi c IgG fl uoride (PMSF) as protein inhibitors. The salivary glands were antibodies have been developed as serological indicators or stored at -80°C until use. biomarkers of exposure to malaria vectors(14). Salivary gland extraction and protein quantifi cation Immunoglobulin G antibodies against Anopheles salivary proteins in the host may be associated with protection against One hundred salivary glands pairs in PBS and PMSF were malaria. In malaria-endemic regions, populations exposed thawed on ice and mixed in 1:1 lysis buffer containing 1.5mM to uninfected Anopheles bites repeatedly over the years may MgCl2, 10mM Tris HCl, 10mM NaCl, 1% Nonidet P-40, and develop an anti-saliva immune response by producing specifi c 2mM ethylenediaminetetraacetic acid (EDTA) NaOH(20). The antibodies in the presence of interleukin (IL)-10. These specifi c mixture was homogenized using a micropestle and sonicated antibodies will neutralize some salivary proteins of vectors, using a water sonicator for 30 min. After centrifugation thus causing micro-environmental changes at the site of the (12,600rpm for 15 min at 4°C), the resulting supernatant was mosquito bite and ultimately affecting malaria transmission. collected and concentrated using a spin concentrator (cut-off of Therefore, in asymptomatic patients with malaria, the level of 10kDa; Corning) and centrifugation (10,000rpm for 30s at 4°C). IgG antibodies targeting anti-salivary gland sonicates (SGSs) The protein concentrations of SGEs were determined using a from Anopheles darlingi are higher than those in symptomatic nanophotometer (Nanophotometer Implen P 360, Germany). patients and healthy individuals(15). Exposure to Anopheles Salivary gland extracts were then diluted in 0.1M bicarbonate mosquito bites and Plasmodium infections, which persist for a buffer (pH 9.6) to obtain a protein concentration of 1µg/µL for long time, affect the development of the immune response to enzyme-linked immunosorbent assay (ELISA)(11). parasites and salivary vectors, thereby infl uencing the number Human serum samples of parasites and the host response(16). Thus, salivary components could be effective vaccine candidates for reducing the morbidity Twenty serum samples were collected from healthy of vector-borne diseases through combination with other malaria adult residents living in the location at which we collected vaccine candidates to protect against severe malaria(7). Further An. sundaicus, i.e., Bangsring village, Wongsorejo District, characterization of salivary proteins and the immune response Banyuwangi Regency in East Java province, to detect antibodies is needed to identify salivary proteins involved in protection against An. sundaicus salivary gland proteins. Seven serum against malaria; the fi rst step in this process is determination samples from healthy adult residents living in non-malaria of the immunogenicity of the salivary proteins. Many studies regions were used as negative control. The human subjects have been conducted to identify and characterize immunogenic protocol for this study was approved by the Ethical Committee salivary proteins from malaria vectors in Africa; however, of Medical Research, Medical Faculty, Brawijaya University. malaria vectors in Asia, particularly An. sundaicus, have not ELISA been studied(8) (17) (18). Therefore, in this study, we measured the levels of IgG To optimize the working conditions for ELISAs, checkerboard antibodies targeting salivary proteins in serum samples from titration was carried out using An. sundaicus SGEs at 1, 2, and individuals living in malaria-endemic areas using salivary 4µg/mL and serially diluted serum samples (1:25, 1:50, and glands extracts (SGEs) from An. sundaicus. Based on the 1:100) from healthy individuals living in the malaria-endemic IgG antibody response to salivary proteins, we identifi ed the region. ELISA was performed as described by Fontaine immunogenic proteins contained within An. sundaicus SGEs. et al.(11). Based on the results of checkerboard titration, microtiter immunoplates (SPL Life Sciences, Korea) were coated with 4µg/ml (50µL/well) of An. sundaicus SGEs diluted in 0.1M METHODS bicarbonate buffer (pH 9.6) overnight at 4°C. Three washes were carried out using PBS-T (PBS, pH 7.4, containing 0.05% Anopheles mosquitoes and isolation of salivary glands Tween-20; Nacalai, Japan) between each incubation. The plates The adult female Anopheles sundaicus mosquitoes used in were blocked for 2h at 37°C with 200µL of blocking solution this study were collected from Bangsring village, Wongsorejo buffer consisting of PBS, 0.05% Tween-20, and 1% bovine District, Banyuwangi Regency in East Java province using serum albumin (BSA; SERVA, Germany). Serum diluted 1:25 aspirators. In this area, Anopheles sundaicus is the dominant in blocking buffer was added (50µL/well) into duplicate wells vector because its population is larger than that of other and incubated at 37°C for 1h. After washing, 50µL of horse common species, such as Anopheles vagus, Anopheles subpictus, radish peroxidase (HRP)-conjugated rabbit anti-human IgG Anopheles barbirostris, and Anopheles annularis(19). These (1:5,000; Surmodics, USA) diluted in blocking buffer was added, mosquitoes were maintained in the insectariums of the Zoology and the plates were incubated at 37°C for 1h. Enzyme activity Laboratory of Biology Department, Faculty of Mathematic was detected by incubation with 50µL of tetramethylbenzidine and Natural Sciences, Jember University at 28°C with substrate (KPL, USA) for 30 min at room temperature. Fifty

60-70% relative humidity and 10% sucrose. The salivary microliters of 1M H2SO4 was added to stop the reaction.

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The optical density (OD) at 450nm was determined with a microplate reader (Bio-Rad, USA). A pool of fi ve serum samples A B from individuals living in Bangsring village, which all exhibited high levels of antibody responses against An. sundaicus SGEs based on the ELISA optimization test, was used as a positive control. The negative control was individual serum samples from individuals who had never been exposed to An. sundaicus bites. The level of IgG antibodies was expressed as the adjusted OD (aOD), which was calculated for each serum sample duplicate as the mean OD value for wells with SGEs minus the OD value of the control wells, i.e., without SGEs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis FIGURE 1 - Single salivary glands from adult Anopheles Sodium dodecyl sulfate polyacrylamide gel electrophoresis sundaicus mosquitoes. A) A female salivary gland. B) A male salivary gland. DL: distal region of lateral lobe; PL: proximal (SDS-PAGE) was performed according to the methods described region of the lateral lobe; ML: median lobe. (Nikon stereoscopic by Jariyapan et al.(21). Briefl y, ten salivary gland pairs were microscope at 4× magnifi cation). mixed 1:2 in 1× SDS gel loading buffer and boiled in a water bath for 5 min. The mixtures were then loaded on 12% SDS polyacrylamide gels. To visualize the bands, gels were stained with Coomassie Brilliant Blue (CBB). A molecular weight ranging from 24 to 138kDa (Figure 2). Among these 16 bands, marker (Nacalai) was loaded on each gel to identify the proteins there were some major bands observed at estimated molecular in SGEs. weights of 144, 120, 91, 62, 46, 40, 36, 33, 31, 26, and 22kDa. Western blotting of salivary gland proteins Levels of anti-salivary gland protein IgG antibodies Gels from SDS-PAGE were transferred to polyvinylidene ELISA was used to detect and measure the levels of anti- difluoride (PVDF) membranes (MACHEREY-NAGEL, salivary gland protein IgG antibodies. The levels of antibodies Germany) using semidry blotting (Bio-Rad) for 1h at 100mA. against salivary proteins in sera from healthy individuals living The membranes were blocked by incubation in 5% nonfat dry in Bangsring village were variable. These variations could be milk dissolved in (blocking buffer) for 1h at room temperature. infl uenced by the intensity of exposure to mosquito bites and After washing with -Tween 0.05% (T) three times, membranes mosquito density(11). There were three serum samples with low were incubated overnight at 4°C with serum samples diluted OD values (less than that of the negative control) among the 20 1:20 in blocking buffer. Subsequently, membranes were serum samples. Therefore, these three samples were not used incubated with alkaline-phosphatase goat anti-human IgG for detection of immunogenic proteins by western blotting. secondary antibodies at a dilution of 1:2,000 for 2h at room temperature after three washes in TBST. Nitro blue tetrazolium- Immunogenic proteins found in Anopheles sundaicus bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) Phosphatase salivary glands substrate was used for color development. To estimate the The results of western blotting showed the presence of protein size, prestained broad-range molecular weight markers several immunogenic proteins with molecular masses of 56, 46, (9-200 kDa; Nacalai) were used. 41, 38, 36, 33, 31, and 26kDa (Figure 3). These results were obtained by using sera from individuals living in Bangsring RESULTS village, and three repetitions were performed. Among the protein bands recognized by the anti-salivary protein antibody, the most Protein profi les of Anopheles sundaicus immunogenic proteins had molecular masses of 46, 41, 33, and salivary glands 31kDa (these protein bands were present in 10-12 serum samples). These immunogenics proteins were consistently recognized by Salivary glands of female mosquitoes consist of three lobes: individual responses from 17 serum samples and by pooled serum the two lateral lobes and the medial lobe, which is attached to from individuals living in malaria-endemic area (Bangsring the salivary duct (Figure 1). The lateral lobes are longer than village) as a positive control (Figure 4A). The human antibody medial lobe and are formed by the proximal, intermediate, and response to salivary proteins from female Anopheles mosquitoes distal regions, whereas the median lobe is formed by a short neck is also specifi c to Anopheles mosquito bites(12) (17). Our result region and distal region. Based on the results of SDS-PAGE, we showed that immunogenic proteins were not detected by western found that the protein profi les of salivary glands from female blotting in pooled serum samples from seven individuals living An. sundaicus consisted of 16 bands with molecular weights in non-malaria-endemic areas as a negative control (Figure 4B).

412 Armiyanti Y et al. - Anopheles immunogenic protein

kDa M kDa M An. sundaiucus 200 120 91 200 62 46 46 41 144 38 33 31 120 120 26

91 91 9

FIGURE 3 - Western blotting using Anopheles sundaicus salivary 70 gland proteins detected with human sera from individuals 62 living in a malaria-endemic area. The most highly immunogenic 62 proteins are shown by intense bands at 46, 41, 33, and 31kDa. 56 kDa: Kilodalton; M: Marker. 52

46 46

41 40 38 38 kDa M 36 kDa M

33 200 200 120 31 120

91 91 26 26 62 62

46 46 46 22 41 38

38 19 31

19 9 9 A B 9

FIGURE 2 - Salivary gland proteins from female Anopheles FIGURE 4 - A) Western blotting of anti-salivary gland protein sundaicus mosquitoes, separated using 12% SDS-PAGE (right lane) IgG antibodies in a pool of human serum samples from and stained with Coomassie Blue. Molecular weight markers are individuals living in a malaria-endemic area (positive control)) shown in the left lane. kDa: Kilodalton; M: Marker; An.: Anopheles; and B) individuals living in a non-malaria endemic area (negative SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. control). kDa: Kilodalton; M: Marker; IgG: immnoglobulin G.

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a short time. Consistent with this, in a study in Senegal, children DISCUSSION who experienced low and moderate exposure exhibited decreased antibody concentrations clearly after more than 3 months(10). The protein profiles of Anopheles sundaicus salivary Another study showed that the IgG antibody response to salivary glands have not been studied before; however, some studies gland proteins appearing after exposure to mosquito bites lasts 3-6 have reported the protein profi les of other Anopheles species, weeks(8). Although the duration of the IgG response to salivary gland i.e., Anopheles dirus, Anopheles gambiae, and Anopheles proteins may be brief (between 1 and 4 months), this response can stephensi(12) (17) (18) (21). A previous study by Cornelie et al.(17) detect specifi c proteins of the salivary gland, i.e., gSG6, in children demonstrated the expression of 20 proteins from the salivary exposed to very infrequent Anopheles bites. According to this study, glands of An. gambiae, which were clearly detected in immunogenic proteins from the salivary glands of Anopheles could silver stained gels. Some of these proteins had the be developed as immunoepidemiological markers to assess the same molecular weights as proteins from salivary glands of risk to very infrequent Anopheles bites in the context of changes Anopheles sundaicus, i.e., 61-63, 52-54, 41-44, 38-39, 31-34, in seasons, different environmental conditions, and travel(13). and 26kDa, similar to the major protein bands detected from The observed immunogenic proteins with molecular the salivary glands of An. dirus(21). This result may be explained weights of 41 and 46kDa could be members of the SG1 family by the presence of several proteins in the salivary glands or TRIO proteins, which have molecular weights ranging of Anopheles that are conserved at the genus level(7). Some from 40 to 48kDa(23) (24). TRIO is a multidomain protein that proteins families are found in all Anopheles species; these are binds the lymphocyte activating receptor transmembrane called genus-specifi c anopheline secreted proteins and include tyrosine phosphatase (PTPase) and contains a protein kinase apyrase/5′ nucleotidase, antigen 5/gvag, GE-rich/30kDa, long domain; however, the function of this protein still needs to and short form D7, mucin/13.5kDa, SG3, SG7, SG10, and be determined(22). Our results also showed that proteins with hypothetical 6.2-kDa protein families(18). Potential proteins molecular weights of 41 and 46kDa were the most immunogenic having molecular weights of 61-63kDa include apyrase/5′ because these proteins were recognized by anti-salivary gland nucleotidase, which is involved in the blood feeding process protein antibodies from 12 serum samples. According to a through degradation (hydrolysis) of adenosine diphosphate previous study, TRIO protein is antigenic in four species (ADP) and adenosine triphosphate (ATP) to adenosine Anopheles, i.e., Anopheles gambiae, Anopheles arabiensis, monophosphate (AMP), a mediator of platelet aggregation, Anopheles stephensi, and Anopheles albimanus; the results infl ammation, and neutrophil activation(5) (22 (23). Long form D7 of mass spectrometric analyses showed that this protein is protein families have molecular weights ranging from 33 to conserved within the Cellia subgenus(18). TRIO protein is one 34kDa, and GE-rich/anti-platelet family proteins have molecular of several antigenic proteins, in addition to gSG6, gSG1b, SG5, weights around 30kDa; these proteins are also involved in the and long form D7, that are over expressed in salivary glands blood feeding process(22) (23). infected with Plasmodium falciparum. Therefore, TRIO proteins The salivary gland proteins of An. sundaicus could be may be involved in malaria transmission and may represent a (25) recognized by IgG antibodies in human sera from individuals living new candidate biomarker for infected Anopheles bites . in Bangsring village, particularly proteins with molecular masses The observed immunogenic protein with a molecular weight of 56, 46, 41, 38, 36, 33, 31, and 26kDa. Among these proteins, of 33 kDa could be a long form D7 family protein; these proteins those with molecular masses of 46, 41, 33, and 31kDa were the range in molecular weight from 33 to 36 kDa(23). D7 proteins most immunogenic. This result showed that exposure to mosquito are also members of the odorant-binding protein superfamily bites in individuals living in malaria-endemic areas, could induce (ODP) and are found in the salivary glands of blood-sucking the immune response by producing salivary protein-specifi c IgG insects, such as mosquitoes, sand flies, and Culicoides(26). antibodies(10) (12). Indeed, the IgG antibody response against salivary This protein has two types, i.e., the short form, which is only gland proteins can be infl uenced by exposure to mosquito bites. found in mosquitoes, and the long form, which can be found In individuals with different levels of exposure to An. gambiae in mosquitoes and sand fl ies(23) (24). D7 proteins have functions bites, the levels of anti-salivary gland protein IgG antibodies related to the binding of one or more agonists hemostasis and are positively related to the intensity of exposure to Anopheles the blood feeding process(26). Some D7 proteins, i.e., D7r1, mosquito bites. The IgG response increases signifi cantly with the D7r2, D7r3, D7r4, and long form D7, have been shown to bind increase in Anopheles exposure, as evaluated using conventional with biogenic amines compounds, such as serotonin, histamine, and entomological methods during the transmission season(10). The norepinephrine, thus becoming antagonistic to vasoconstrictors results of a study conducted by Fontaine et al. also demonstrated and affecting platelet aggregation and induction of pain(27). a positive correlation between the average IgG response to SGEs According to a previous study(26), which analyzed the salivary of Aedes caspius and Aedes caspius density, which was affected by glands of An. darlingi by mass spectrometry, the 31-kDa protein changes in the season and the ecological environment(18). The level identifi ed in our study could be a member of the 30-kDa allergen of the IgG response increased signifi cantly during peak exposure protein family or the GE-rich/anti-platelet family. Members of to Ae. caspius in September and declined to baseline values the 30-kDa allergen protein family from the salivary glands of within 4 months (January)(11). These results also showed that IgG Ae. aegypti were shown to be associated with an allergic reaction responses induced by mosquito saliva antigens persisted for only to mosquito bites involving IgE and lymphocyte-mediated

414 Armiyanti Y et al. - Anopheles immunogenic protein hypersensitivity(28). This protein, also called aegyptin, has been shown to inhibit platelet aggregation induced by collagen in ACKNOWLEDGMENTS humans and to impede granule secretion. Aegyptin recognizes the speciﬁ c binding sites of glycoprotein IV, integrin α2β1, and The authors would like to thank all the laboratory staff of von Willebrand factor and can the refore prevent the interaction the Biology Laboratory, Biology Department, Mathematic and between collagen and these major ligands(29). Anophelin anti- Natural Sciences Faculty, Jember University. This study was platelet protein (AAPP) is a protein found in the salivary glands funded by a Doctorate Research Grant of the Directorate General of An. stephensi that is homologous to the 30-kDa Aedes aegypti of Higher Education Indonesia. allergen(23). This protein is also a member of the GE-rich family of proteins. AAPP has been shown to block the adhesion of platelets CONFLICT OF INTEREST to collagen via direct binding to collagen, subsequently inhibiting the increase in intracellular calcium ion concentration(30). The authors declare that there is no conﬂ ict of interest. Therefore, this protein has an important role in blood feeding by inhibition of the hemostatic process. FINANCIAL SUPPORT This study is the first to show the protein profile and immunogenic proteins from the salivary glands of This study was funded by a Doctorate Research Grant of the An. sundaicus, one of the major malaria vectors in South Asian Directorate General of Higher Education Indonesia. countries. Several studies have described anti-salivary protein antibody responses that recognize some proteins of Anopheles REFERENCES salivary glands, particularly malaria vectors in Africa(17) (18).

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